Actinobacillus pleuropneumoniae outer membrane lipoprotein A and uses thereof

ABSTRACT

Novel vaccines for use against Actinobacillus pleuropneumoniae are disclosed. The vaccines contain at least one Actinobacillus pleuropneumoniae outer membrane lipoprotein A, or an immunogenic fragment thereof. Also disclosed are DNA sequences encoding these proteins, vectors including these sequences and host cells transformed with these vectors. The vaccines can be used to treat or prevent porcine respiratory infections.

TECHNICAL FIELD

The instant invention relates generally to the prevention of disease inswine. More particularly, the present invention relates to subunitvaccines for Actinobacillus pleuropneumoniae.

BACKGROUND

Actinobacillus (formerly Haemophilus) pleuropneumoniae is a highlyinfectious porcine respiratory tract pathogen that causes porcinepleuropneumonia. Infected animals develop acute fibrinous pneumoniawhich leads to death or chronic lung lesions and reduced growth rates.Infection is transmitted by contact or aerosol and the morbidity insusceptible groups can approach 100%. Persistence of the pathogen inclinically healthy pigs also poses a constant threat of transmittingdisease to previously uninfected herds.

The rapid onset and severity of the disease often causes losses beforeantibiotic therapy can become effective. Presently available vaccinesare generally composed of chemically inactivated bacteria combined withoil adjuvants. However, whole cell bacterins and surface proteinextracts often contain immunosuppressive components which make pigs moresusceptible to infection. Furthermore, these vaccines may reducemortality but do not reduce the number of chronic carriers in a herd.

There are at least 12 recognized serotypes of A. pleuropneumoniae withthe most common in North America being serotypes 1, 5 and 7. Differencesamong serotypes generally coincide with variations in theelectrophoretic mobility of outer membrane proteins and enzymes, thusindicating a clonal origin of isolates from the same serotype. Thisantigenic variety has made the development of a successful vaccinationstrategy difficult. Protection after parenteral immunization with akilled bacterin or cell free extract is generally serotype specific anddoes not prevent chronic or latent infection. Higgins, R., et al., Can.Vet. J. (1985) 26:86-89; MacInnes, J. I. and Rosendal, S., Infect.Immun. (1987) 55:1626-1634. Thus, it would be useful to develop vaccineswhich protect against both death and chronicity and do not haveimmunosuppressive properties. One method by which this may beaccomplished is to develop subunit antigen vaccines composed of specificproteins in pure or semi-pure form.

An increasing number of bacterial antigens have now been identified aslipoproteins (Anderson, B. E., et al., J. Bacteriol. (1988)70:4493-4500; Bricker, T. M., et al., Infect. Immun. (1988) 56:295-301;Hanson, M. S., and Hansen, E. J., Mol. Microbiol. (1991) 5:267-278;Hubbard, C. L., et al., Infect. Immun. (1991) 59:1521-1528; Nelson, M.B., et al., Infect. Immun. (1988) 56:128-134; Thirkell, D., et al.,Infect. Immun. (1991) 59:781-784). One such lipoprotein from Haemophilussomnus has been positively identified. The nucleotide sequence for thislipoprotein, termed "LppA," has been determined (Theisen, M., et al.,Infect. Immun. (1992) 60:826-831). These lipoproteins are generallylocalized in the envelope of the cell and are therefore exposed to thehost's immune system. It has been shown that the murine lipoprotein fromthe outer membrane of Escherichia coli acts as a potent activator ofmurine lymphocytes, inducing both proliferation and immunoglobulinsecretion (Bessler, W., et al., Z. Immun. (1977) 153:11-22; Melchers,F., et al., J. Exp. Med. (1975) 142:473-482). The active lipoproteinportion of the protein has been shown to reside in the N-terminal fattyacid containing region of the protein. Recent studies using syntheticlipopeptides based on this protein show that even short peptides,containing two to five amino acids covalently linked to palmitate, areable to activate murine lymphocytes (Bessler, W. G., et al., J. Immunol.(1985) 35:1900-1905).

It has been found that A. pleuropneumoniae possesses several outermembrane proteins which are expressed only under iron limiting growthconditions (Deneer, H. G., and Potter, A. A., Infect. Immun. (1989)57:798-804). However, outer membrane lipoproteins from A.pleuropneumoniae have not heretofore been identified or characterizedwith respect to their immunogenic or protective capacity.

DISCLOSURE OF THE INVENTION

The present invention is based on the discovery of a novel subunitantigen from A. pleuropneumoniae which shows protective capability inpigs.

Accordingly, in one embodiment, the subject invention is directed topurified, immunogenic A. pleuropneumoniae outer membrane lipoprotein A,or an immunogenic fragment thereof.

In another embodiment, the instant invention is directed to an isolatednucleotide sequence comprising a sequence encoding an immunogenic A.pleuropneumoniae outer membrane lipoprotein A, or an immunogenicfragment thereof.

In yet another embodiment, the subject invention is directed to a DNAconstruct comprising the above nucleotide sequence and control sequencesthat are operably linked to the nucleotide sequence whereby thenucleotide sequence can be transcribed and translated in a host cell,and at least one of the control sequences is heterologous to thenucleotide sequence.

In still further embodiments, the instant invention is directed to hostcells transformed with these constructs and methods of recombinantlyproducing the subject A. pleuropneumoniae proteins.

In another embodiment, the subject invention is directed to a vaccinecomposition comprising a pharmaceutically acceptable vehicle and an A.pleuropneumoniae outer membrane lipoprotein A or an immunogenic fragmentthereof.

In still another embodiment, the invention is directed to a method oftreating or preventing an A. pleuropneumoniae infection in a vertebratesubject comprising administering to the subject a therapeuticallyeffective amount of a vaccine composition as described above.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E depict the nucleotide sequence of the gene coding for A.pleuropneumoniae serotype 1 outer membrane lipoprotein A as well as thenucleotide sequence for the flanking regions from the HB101/pOM37/E16clone (SEQ ID: 1). The predicted amino acid sequence is also shown.

FIGS. 2A-2G depict the nucleotide sequence of the gene encoding for A.pleuropneumoniae serotype 5 outer membrane lipoprotein A as well as thenucleotide sequence for the flanking regions from HB101/pSR213/E25 (SEQID: 2). The predicted amino acid sequence is also shown.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,virology, recombinant DNA technology, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II(D. N. Glover, ed., 1985); Oligonucleotide synthesis (M. J. Gait, ed.,1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds.,1984); Animal Cell Culture (R. K. Freshney, ed., 1986); ImmobilizedCells and Enzymes (IRL press, 1986); Perbal, B., A Practical Guide toMolecular Cloning (1984); the series, Methods In Enzymology (S. Colowickand N. Kaplan, eds., Academic Press, Inc.); and Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986,Blackwell Scientific Publications).

All patents, patent applications and publications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

A. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms "outer membrane lipoprotein A" and "OmlA" are equivalent andinterchangeable and define a protein from the family of proteinsrepresented by A. pleuropneumoniae serotype 1 OmlA (depicted in FIG. 1)and A. pleuropneumoniae serotype 5 OmlA (depicted in FIG. 2). The term"OmlA" also captures proteins substantially homologous and functionallyequivalent to native OmlAs. Thus, the term encompasses modifications,such as deletions, additions and substitutions (generally conservativein nature), to the native sequences, as long as immunological activity(as defined below) is not destroyed. Such modifications of the primaryamino acid sequence may result in antigens which have enhanced activityas compared to the native sequence. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts which produce the lipoprotein. All ofthese modifications are included, so long as immunogenic activity isretained. Accordingly, A. pleuropneumoniae serotype 1 OmlA and A.pleuropneumoniae serotype 5 OmlA refer not only to the amino acidsequences depicted in FIGS. 1 and 2, repectively, but to amino acidsequences homologous thereto which retain the defined immunologicalactivity.

Additionally, the term "OmlA" (or fragments thereof) denotes a proteinwhich occurs in neutral form or in the form of basic or acid additionsalts, depending on the mode of preparation. Such acid salts may involvefree amino groups and basic salts may be formed with free carboxyls.Pharmaceutically acceptable basic and acid addition salts are discussedfurther below. In addition, the protein may be modified by combinationwith other biological materials such as lipids (either those normallyassociated with the lipoprotein or other lipids that do not destroyactivity) and saccharides, or by side chain modification, such asacetylation of amino groups, phosphorylation of hydroxyl side chains, oroxidation of sulfhydryl groups, as well as other modifications of theencoded primary sequence. Thus, included within the definition of "OmlA"herein are glycosylated and unglycosylated forms, the amino acidsequences with or without associated lipids, and amino acid sequencessubstantially homologous to the native sequence which retain the abilityto elicit an immune response.

Two DNA or polypeptide sequences are "substantially homologous" when atleast about 65% (preferably at least about 80% to 90%, and mostpreferably at least about 95%) of the nucleotides or amino acids matchover a defined length of the molecule. As used herein, substantiallyhomologous also refers to sequences showing identity to the specifiedDNA or polypeptide sequence. DNA sequences that are substantiallyhomologous can be identified in a Southern hybridization experimentunder, for example, stringent conditions, as defined for that particularsystem. Defining appropriate hybridization conditions is within theskill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, vols I& II, supra; Nucleic Acid Hybridiation, supra.

The term "functionally equivalent" intends that the amino acid sequenceof the subject protein is one that will elicit an immunologicalresponse, as defined below, equivalent to or better than, theimmunological response elicited by a native A. pleuropneumoniae OmlA.

An "antigen" refers to a molecule containing one or more epitopes thatwill stimulate a host's immune system to make a humoral and/or cellularantigen-specific response. The term is also used interchangeably with"immunogen."

By "subunit antigen" is meant an antigen entity separate and discretefrom a whole bacterium (live or killed). Thus, an antigen contained in acell free extract would constitute a "subunit antigen" as would asubstantially purified antigen.

A "hapten" is a molecule containing one or more epitopes that does notstimulate a host's immune system to make a humoral or cellular responseunless linked to a carrier.

The term "epitope" refers to the site on an antigen or hapten to which aspecific antibody molecule binds. The term is also used interchangeablywith "antigenic determinant" or "antigenic determinant site."

An "immunological response" to an antigen or vaccine is the developmentin the host of a cellular and or antibody-mediated immune response tothe composition or vaccine of interest. Usually, such a responseincludes but is not limited to one or more of the following effects; theproduction of antibodies, B cells, helper T cells, suppressor T cells,and/or cytotoxic T cells and/or γδ T cells, directed specifically to anantigen or antigens included in the composition or vaccine of interest.

The terms "immunogenic polypeptide" and "immunogenic amino acidsequence" refer to a polypeptide or amino acid sequence, respectively,which elicit antibodies that neutralize bacterial infectivity, and/ormediate antibody-complement or antibody dependent cell cytotoxicity toprovide protection of an immunized host. An "immunogenic polypeptide" asused herein, includes the full length (or near full length) sequence ofan A. pleuropneumoniae OmlA, or an immunogenic fragment thereof. By"immunogenic fragment" is meant a fragment of an A. pleuropneumoniaeOmlA which includes one or more epitopes and thus elicits antibodiesthat neutralize bacterial infectivity, and/or mediateantibody-complement or antibody dependent cell cytotoxicity to provideprotection of an immunized host. Such fragments will usually be at leastabout 5 amino acids in length, and preferably at least about 10 to 15amino acids in length. There is no critical upper limit to the length ofthe fragment, which could comprise nearly the full length of the proteinsequence, or even a fusion protein comprising fragments of two or moreof the A. pleuropneumoniae subunit antigens.

The terms "polypeptide" and protein are used interchangeably and referto any polymer of amino acids (dipeptide or greater) linked throughpeptide bonds. Thus, the terms "polypeptide" and "protein" includeoligopeptides, protein fragments, analogs, muteins, fusion proteins andthe like.

"Native" proteins or polypeptides refer to proteins or polypeptidesrecovered from a source occurring in nature. Thus, the term "nativeouter membrane lipoprotein A" would include naturally occurring OmlA andfragments of these proteins.

By "purified protein" is meant a protein separate and discrete from awhole organism (live or killed) with which the protein is normallyassociated in nature. Thus, a protein contained in a cell free extractwould constitute a "purified protein," as would a protein syntheticallyor recombinantly produced.

"Recombinant" polypeptides refer to polypeptides produced by recombinantDNA techniques; i.e., produced from cells transformed by an exogenousDNA construct encoding the desired polypeptide. "Synthetic" polypeptidesare those prepared by chemical synthesis.

A "replicon" is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A "vector" is a replicon, such as a plasmid, phage, or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A "double-strandedDNA molecule" refers to the polymeric form ofdeoxyribonucleotides (bases adenine, guanine, thymine, or cytosine) in adouble-stranded helix, both relaxed and supercoiled. This term refersonly to the primary and secondary structure of the molecule, and doesnot limit it to any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5' to 3' direction along thenontranscribed strand of DNA (i.e., the strand having the sequencehomologous to the mRNA).

A DNA "coding sequence" or a "nucleotide sequence encoding" a particularprotein, is a DNA sequence which is transcribed and translated into apolypeptide in vivo or in vitro when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5' (amino) terminus and atranslation stop codon at the 3' (carboxy) terminus. A coding sequencecan include, but is not limited to, procaryotic sequences, cDNA fromeucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian)DNA, and even synthetic DNA sequences. A transcription terminationsequence will usually be located 3' to the coding sequence.

A "promoter sequence" is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3'direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bound at the 3' terminus by thetranslation start codon (ATG) of a coding sequence and extends upstream(5' direction) to include the minimum number of bases or elementsnecessary to initiate transcription at levels detectable abovebackground. Within the promoter sequence will be found a transcriptioninitiation site (conveniently defined by mapping with nuclease S1), aswell as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. Eucaryotic promoters will often, but notalways, contain "TATA" boxes and "CAT" boxes. Procaryotic promoterscontain Shine-Dalgarno sequences in addition to the -10 and -35consensus sequences.

DNA "control sequences" refers collectively to promoter sequences,ribosome binding sites, polyadenylation signals, transcriptiontermination sequences, upstream regulatory domains, enhancers, and thelike, which collectively provide for the transcription and translationof a coding sequence in a host cell.

"Operably linked" refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered "operably linked" to thecoding sequence.

A control sequence "directs the transcription" of a coding sequence in acell when RNA polymerase will bind the promoter sequence and transcribethe coding sequence into mRNA, which is then translated into thepolypeptide encoded by the coding sequence.

A "host cell" is a cell which has been transformed, or is capable oftransformation, by an exogenous DNA sequence.

A cell has been "transformed" by exogenous DNA when such exogenous DNAhas been introduced inside the cell membrane. Exogenous DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In procaryotes and yeasts, for example, theexogenous DNA may be maintained on an episomal element, such as aplasmid. With respect to eucaryotic cells, a stably transformed cell isone in which the exogenous DNA has become integrated into the chromosomeso that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeucaryotic cell to establish cell lines or clones comprised of apopulation of daughter cell containing the exogenous DNA.

A "clone" is a population of cells derived from a single cell or commonancestor by mitosis. A "cell line" is a clone of a primary cell that iscapable of stable growth in vitro for many generations.

A "heterologous" region of a DNA construct is an identifiable segment ofDNA within or attached to another DNA molecule that is not found inassociation with the other molecule in nature. Thus, when theheterologous region encodes a bacterial gene, the gene will usually beflanked by DNA that does not flank the bacterial gene in the genome ofthe source bacteria. Another example of the heterologous coding sequenceis a construct where the coding sequence itself is not found in nature(e.g., synthetic sequences having codons different from the nativegene). Allelic variation or naturally occurring mutational events do notgive rise to a heterologous region of DNA, as used herein.

A composition containing A is "substantially free of" B when at leastabout 85% by weight of the total of A+B in the composition is A.Preferably, A comprises at least about 90% by weight of the total of A+Bin the composition, more preferably at least about 95%, or even 99% byweight.

The term "treatment" as used herein refers to either (i) the preventionof infection or reinfection (prophylaxis), or (ii) the reduction orelimination of symptoms of the disease of interest (therapy).

B. General Methods

Central to the present invention is the discovery of a family of A.pleuropneumoniae outer membrane lipoproteins, termed OmlAs herein, whichare able to elicit an immune response in an animal to which they areadministered. All 12 of the A. pleuropneumoniae serotypes appear tocontain a gene encoding an OmlA. This protein, analogs thereof and/orimmunogenic fragments derived from the protein, are provided in subunitvaccine compositions and thus problems inherent in prior vaccinecompositions, such as localized and systemic side reactions, as well asthe inability to protect against chronic disease, are avoided. Thevaccine compositions can be used to treat or prevent A.pleuropneumoniae-induced respiratory diseases in swine such as porcinepleuropneumonia. The antigens or antibodies thereto can also be used asdiagnostic reagents to detect the presence of an A. pleuropneumoniaeinfection in a subject. Similarly, the genes from the various serotypesencoding the OmlA proteins can be cloned and used to design probes forthe detection of A. pleuropneumoniae in tissue samples as well as forthe detection of homologous genes in other bacterial strains. Thesubunit antigens can be conveniently produced by recombinant techniques,as described herein. The proteins of interest are produced in highamounts in transformants, do not require extensive purification orprocessing, and do not cause lesions at the injection site or other illeffects.

The genes encoding the A. pleuropneumoniae serotype 1 OmlA and serotype5 OmlA have been isolated and the sequences are depicted in FIG. 1 (SEQID NO: 1) and FIG. 2 (SEQ ID NO: 2), respectively. The nucleotidesequence for the serotype 1 omlA gene, including the structural gene andflanking regions, consists of approximately 1340 base pairs. The openreading frame codes for a protein having approximately 365 amino acids.The nucleotide sequence for the serotype 5 omlA gene, including thestructural gene and flanking regions, consists of approximately 2398base pairs. The structural gene codes for a protein of approximately 367amino acidS. The serotype 1 and serotype 5 OmlA proteins areapproximately 65% homologous.

The omlA gene from A. pleuropneumoniae serotype 1 hybridizes withgenomic DNA from all other known A. pleuropneumoniae serotypes. Theinvention, therefore, encompasses genes encoding OmlA from all of the A.pleuropneumoniae serotypes.

The full-length serotype 1 and serotype 5 lipoproteins both have anapparent molecular mass of approximately 50 kDa, as determined bydiscontinuous sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) according to the method of Laemmli (Laemmli, M. K., Nature(1970) 227:680-685). The predicted molecular weights, based on the aminoacid sequences, are 39,780 and 40,213, respectively. The recombinantlyproduced proteins are able to protect pigs from subsequent challengewith A. pleuropneumoniae. Other OmlA proteins, from other A.pleuropneumoniae serotypes, can also be identified, purified andsequenced, using any of the various methods known-to those skilled inthe art. For example, the amino acid sequences of the subject proteinscan be determined from the purified proteins by repetitive cycles ofEdman degradation, followed by amino acid analysis by HPLC. Othermethods of amino acid sequencing are also known in the art. Fragments ofthe purified proteins can be tested for biological activity and activefragments, as described above, used in compositions in lieu of theentire protein.

In order to identify genes encoding the subject proteins, recombinanttechniques can be employed. For example a DNA library can be preparedwhich consists of genomic DNA from an A. pleuropneumoniae serotype. Theresulting clones can be used to transform an appropriate host, such asE. coli. Individual colonies can then be screened in an immunoblotassay, using polyclonal serum or monoclonal antibodies, to the desiredantigen.

More specifically, after preparation of a DNA library, DNA fragments ofa desired length are isolated by, e.g., sucrose density gradientcentrifugation. These fragments are then ligated into any suitableexpression vector or replicon and thereafter the corresponding host cellis transformed with the constructed vector or replicon. Transformedcells are plated in suitable medium. A replica plate must also beprepared because, subsequent procedures kill these colonies. Thecolonies are then lysed in one of a number of ways, e.g., by exposure tochloroform vapor. This releases the antigen from the positive colonies.The lysed colonies are incubated with the appropriate unlabelledantibody and developed using an appropriate anti-immunoglobulinconjugate and substrate. Positively reacting colonies thus detected canbe recovered from the replica plate and subcultured. Physical mapping,construction of deletion derivatives and nucleotide sequencing can beused to characterize the encoding gene.

An alternative method to identify genes encoding the proteins of thepresent invention, once the genomic DNA library is constructed asdescribed above, is to prepare oligonucleotides to probe the library andto use these probes to isolate the gene encoding the desired protein.The basic strategies for preparing oligonucleotide probes, as well asscreening libraries using nucleic acid hybridization, are well known tothose of ordinary skill in the art.. See, e.g., DNA Cloning: Vol. I,supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis,supra; Sambrook et al., supra. The particular nucleotide sequencesselected are chosen so as to correspond to the codons encoding a knownamino acid sequence from the desired protein. Since the genetic code isdegenerate, it will often be necessary to synthesize severaloligonucleotides to cover all, or a reasonable number of, the possiblenucleotide sequences which encode a particular region of the protein.Thus, it is generally preferred in selecting a region upon which to basethe probes, that the region not contain amino acids whose codons arehighly degenerate. In certain circumstances, one of skill in the art mayfind it desirable to prepare probes that are fairly long, and/orencompass regions of the amino acid sequence which would have a highdegree of redundancy in corresponding nucleic acid sequences,particularly if this lengthy and/or redundant region is highlycharacteristic of the protein of interest. It may also be desirable touse two probes (or sets of probes), each to different regions of thegene, in a single hybridization experiment. Automated oligonucleotidesynthesis has made the preparation of large families of probesrelatively straight-forward. While the exact length of the probeemployed is not critical, generally it is recognized in the art thatprobes from about 14 to about 20 base pairs are usually effective.Longer probes of about 25 to about 60 base pairs are also used.

The selected oligonucleotide probes are labeled with a marker, such as aradionucleotide or biotin using standard procedures. The labeled set ofprobes is then used in the screening step, which consists of allowingthe single-stranded probe to. hybridize to isolated ssDNA from thelibrary, according to standard techniques. Either stringent orpermissive hybridization conditions could be appropriate, depending uponseveral factors, such as the length of the probe and whether the probeis derived from the same species as the library, or an evolutionarilyclose or distant species. The selection of the appropriate conditions iswithin the skill of the art. See, generally, Nucleic Acid hybridization,supra. The basic requirement is that hybridization conditions be ofsufficient stringency so that selective hybridization occurs; i.e.,hybridization is due to a sufficient degree of nucleic acid homology(e.g., at least about 75%), as opposed to nonspecific binding. Once aclone from the screened library has been identified by positivehybridization, it can be confirmed by restriction enzyme analysis andDNA sequencing that the particular library insert contains a gene forthe desired protein.

Alternatively, DNA sequences encoding the proteins of interest can beprepared synthetically rather than cloned. The DNA sequence can bedesigned with the appropriate codons for the particular amino acidsequence. In general, one will select preferred codons for the intendedhost if the sequence will be used for expression. The complete sequenceis assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence. See, e.g., Edge(1981) Nature 292:756; Nambair et al., (1984) Science 223:1299; Jay etal., (1984) J. Biol. Chem. 259:6311.

Once coding sequences for the desired proteins have been prepared orisolated, they can be cloned into any suitable vector or replicon.Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage λ (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, generally, DNA Cloning: Vols. I & II, supra;Sambrook et al., supra; B. Perbal, supra.

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as "control" elements), so that the DNAsequence encoding the desired protein is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. Leader sequences can be removed by the hostin post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739;4,425,437; 4,338,397.

In addition to control sequences, it may be desirable to add regulatorysequences which allow for regulation of the expression of the proteinsequences relative to the growth of the host cell. Regulatory sequencesare known to those of skill in the art, and examples include those whichcause the expression of a gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Other types of regulatory elements may also be present in thevector, for example, enhancer sequences.

An expression vector is constructed so that the particular codingsequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the control sequences being such that the coding sequence istranscribed under the "control" of the control sequences (i.e., RNApolymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence). Modification of the sequences encodingthe particular antigen of interest may be desirable to achieve this end.For example, in some cases it may be necessary to modify the sequence sothat it may be attached to the control sequences with the appropriateorientation; i.e., to maintain the reading frame. The control sequencesand other regulatory sequences may be ligated to the coding sequenceprior to insertion into a vector, such as the cloning vectors describedabove. Alternatively, the coding sequence can be cloned directly into anexpression vector which already contains the control sequences and anappropriate restriction site.

In some cases, it may be desirable to add sequences which cause thesecretion of the polypeptide from the host organism, with subsequentcleavage of the secretory signal. It may also be desirable to producemutants or analogs of the antigens of interest. Mutants or analogs maybe prepared by the deletion of a portion of the sequence encoding theprotein, by insertion of a sequence, and/or by substitution of one ormore nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, are wellknown-to those skilled in the art. See, e.g., Sambrook et al., supra;DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

A number of procaryotic expression vectors are known in the art. See,e.g., U.S. Pat. Nos. 4,440,859; 4,436,815; 4,431,740; 4,431,739;4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; seealso U.K. Patent Applications GB 2,121,054; GB 2,008,123; GB 2,007,675;and European Patent Application 103,395. Yeast expression vectors arealso known in the art. See, e.g., U.S. Pat. Nos. 4,446,235; 4,443,539;4,430,428; see also European Patent Applications 103,409; 100,561;96,491.

Depending on the expression system and host selected, the proteins ofthe present invention are produced by growing host cells transformed byan expression vector described above under conditions whereby theprotein of interest is expressed. The protein is then isolated from thehost cells and purified. If the expression system secretes the proteininto growth media, the protein can be purified directly from the media.If the protein is not secreted, it is isolated from cell lysates. Theselection of the appropriate growth conditions and recovery methods arewithin the skill of the art.

OmlA antigens can also be isolated directly from any of the A.pleuropneumoniae serotypes. This is generally accomplished by firstpreparing a crude extract which lacks cellular components and severalextraneous proteins. The desired antigens can then be further purified,i.e., by column chromatography, HPLC, immunoadsorbent techniques orother conventional methods well known in the art.

The proteins of the present invention may also be produced by chemicalsynthesis such as solid phase peptide synthesis, using known amino acidsequences or amino acid sequences derived from the DNA sequence of thegenes of interest. Such methods are known to those skilled in the art.Chemical synthesis of peptides may be preferable if a small fragment ofthe antigen in question is capable of raising an immunological responsein the subject of interest.

The proteins of the present invention or their fragments can be used toproduce antibodies, both polyclonal and monoclonal. If polyclonalantibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat,horse, pig etc.) is immunized with an antigen of the present invention,or its fragment, or a mutated antigen. Serum from the immunized animalis collected and treated according to known procedures. If serumcontaining polyclonal antibodies is used, the polyclonal antibodies canbe purified by immunoaffinity chromatography, using known procedures.

Monoclonal antibodies to the proteins of the present invention, and tothe fragments thereof, can also be readily produced by one skilled inthe art. The general methodology for making monoclonal antibodies byusing hybridoma technology is well known. Immortal antibody-producingcell lines can be created by cell fusion, and also by other techniquessuch as direct transformation of B lymphocytes with oncogenic DNA, ortransfection with Epstein-Barr virus. See, e.g., M. Schreier et al.,Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodiesand T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies(1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783;4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890.Panels of monoclonal antibodies produced against the antigen ofinterest, or fragment thereof, can be screened for various properties;i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies areuseful in purification, using immunoaffinity techniques, of theindividual antigens which they are directed against.

Animals can be immunized with the compositions of the present inventionby administration of the protein of interest, or a fragment thereof, oran analog thereof. If the fragment or analog of the protein is used, itwill include the amino acid sequence of an epitope which interacts withthe immune system to immunize the animal to that and structurallysimilar epitopes.

If synthetic or recombinant proteins are employed, the subunit antigencan be a single polypeptide encoding one or several epitopes from one ormore OmlAs or two or more discrete polypeptides encoding differentepitopes. The subunit antigen, even though carrying epitopes derivedfrom a lipoprotein, does not require the presence of the lipid moiety.However, if the lipid is present, it need not be a lipid commonlyassociated with the lipoprotein, so long as the appropriate immunologicresponse is elicited.

Prior to immunization, it may be desirable to increase theimmunogenicity of the particular protein, or an analog of the protein,or particularly fragments of the protein. This can be accomplished inany one of several ways known to those of skill in the art. For example,the antigenic peptide may be administered linked to a carrier. Suitablecarriers are typically large, slowly metabolized macromolecules such as:proteins; polysaccharides, such as sepharose, agarose, cellulose,cellulose beads and the like; polymeric amino acids such as polyglutamicacid, polylysine, and the like; amino acid copolymers; and inactivevirus particles. Especially useful protein substrates are serumalbumins, keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art.

The protein substrates may be used in their native form or theirfunctional group content may be modified by, for example, succinylationof lysine residues or reaction with Cys-thiolactone. A sulfhydryl groupmay also be incorporated into the carrier (or antigen) by, for example,reaction of amino functions with 2-iminothiolane or theN-hydroxysuccinimide ester of 3-(4-dithiopyridyl propionate. Suitablecarriers may also be modified to incorporate spacer arms (such ashexamethylene diamine or other bifunctional molecules of similar size)for attachment of peptides.

Other suitable carriers for the proteins of the present inventioninclude VP6 polypeptides of rotaviruses, or functional fragmentsthereof, as disclosed in allowed U.S. Pat. No. 5,071,651 andincorporated herein by reference. Also useful is a fusion product of aviral protein and the subject immunogens made by methods disclosed inU.S. Pat. No. 4,722,840. Still other suitable carriers include cells,such as lymphocytes, since presentation in this form mimics the naturalmode of presentation in the subject, which gives rise to the immunizedstate. Alternatively, the proteins of the present invention may becoupled to erythrocytes, preferably the subject's own erythrocytes.Methods of coupling peptides to proteins or cells are known to those ofskill in the art.

The novel proteins of the instant invention can also be administered viaa carrier virus which expresses the same. Carrier viruses which willfind use with the instant invention include but are not limited to thevaccinia and other pox viruses, adenovirus, and herpes virus. By way ofexample, vaccinia virus recombinants expressing the novel proteins canbe constructed as follows. The DNA encoding the particular protein isfirst inserted into an appropriate vector so that it is adjacent to avaccinia promoter and flanking vaccinia DNA sequences, such as thesequence encoding thymidine kinase (TK). This vector is then used totransfect cells which are simultaneously infected with vaccinia.Homologous recombination serves to insert the vaccinia promoter plus thegene encoding the instant protein into the viral genome. The resultingTK⁻ recombinant can be selected by culturing the cells in the presenceof 5-bromodeoxyuridine and picking viral plaques resistant thereto.

It is also possible to immunize a subject with a protein of the presentinvention, or a protective fragment thereof, or an analog thereof, whichis administered alone, or mixed with a pharmaceutically acceptablevehicle or excipient. Typically, vaccines are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation may also be emulsified or the activeingredient encapsulated in liposome vehicles. The active immunogenicingredient is often mixed with vehicles containing excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable vehicles are, for example, water, saline, dextrose, glycerol,ethanol, or the like, and combinations thereof. In addition, if desired,the vehicle may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, or adjuvants whichenhance the effectiveness of the vaccine. Adjuvants may include forexample, muramyl dipeptides, avridine, aluminum hydroxide, oils,saponins and other substances known in the art. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in the art. See, e.g., Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Penn., 15th edition, 1975. The compositionor formulation to be administered will, in any event, contain a quantityof the protein adequate to achieve the desired immunized state in theindividual being treated.

Additional vaccine formulations which are suitable for other modes ofadministration include suppositories and, in some cases, aerosol,intranasal, oral formulations, and sustained release formulations. Forsuppositories, the vehicle composition will include traditional bindersand carriers, such as, polyalkaline glycols, or triglycerides. Suchsuppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10% (w/w), preferablyabout 1% to about 2%. Oral vehicles include such normally employedexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium, stearate, sodium saccharin cellulose, magnesiumcarbonate, and the like. These oral vaccine compositions may be taken inthe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations, or powders, and contain from about 10% to about95% of the active ingredient, preferably about 25% to about 70%.

Intranasal formulations will usually include vehicles that neithercauseirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

Controlled or sustained release formulations are made by incorporatingthe protein into carriers or vehicles such as liposomes, nonresorbableimpermeable polymers such as ethylenevinyl acetate copolymers andHytrel® copolymers, swellable polymers such as hydrogels, or resorbablepolymers such as collagen and certain polyacids or polyesters such asthose used to make resorbable sutures. The proteins can also bedelivered using implanted mini-pumps, well known in the art.

Furthermore, the proteins (or complexes thereof) may be formulated intovaccine compositions in either neutral or salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the active polypeptides) and which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric, mandelic, and thelike. Salts formed from free carboxyl groups may also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

To immunize a subject, the polypeptide of interest, or animmunologically active fragment thereof, is administered parenterally,usually by intramuscular injection in an appropriate vehicle. Othermodes of administration, however, such as subcutaneous, intravenousinjection and intranasal delivery, are also acceptable. Injectablevaccine formulations will contain an effective amount of the activeingredient in a vehicle, the exact amount being readily determined byone skilled in the art. The active ingredient may typically range fromabout 1% to about 95% (w/w) of the composition, or even higher or lowerif appropriate. The quantity to be administered depends on the animal tobe treated, the capacity of the animal's immune system to synthesizeantibodies, and the degree of protection desired. With the presentvaccine formulations, as little as 0.1 to 100 μg or more, preferably 0.5to 50 μg, more preferably 1.0 to 25 μg, of active ingredient per ml ofinjected solution, should be adequate to raise an immunological responsewhen a dose of 1 to 2 ml per animal is administered. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. The subject isimmunized by administration of the particular antigen or fragmentthereof, or analog thereof, in at least one dose, and preferably twodoses. Moreover, the animal may be administered as many doses as isrequired to maintain a state of immunity to pneumonia.

An alternative route of administration involves gene therapy or nucleicacid immunization. Thus, nucleotide sequences (and accompanyingregulatory elements) encoding the subject proteins can be administereddirectly to a subject for in vivo translation thereof. Alternatively,gene transfer can be accomplished by transfecting the subjects cells ortissues ex vivo and reintroducing the transformed material into thehost. DNA can be directly introduced into the host organism, i.e., byinjection (see International Publication No. WO/90/11092; and Wolff etal., Science (1990) 247:1465-1468). Liposome-mediated gene transfer canalso be accomplished using known methods. See, e.g., Hazinski et al.,Am. J. Respir. Cell Mol. Biol. (1991) 4:206-209; Brigham et al., Am. J.Med. Sci. (1989) 298:278-281; Canonico et al., Clin. Res. (1991)39:219A; and Nabel et al., Science (1990) 249:1285-1288. Targetingagents, such as antibodies directed against surface antigens expressedon specific cell types, can be covalently conjugated to the liposomalsurface so that the nucleic acid can be delivered to specific tissuesand cells susceptible to A. pleuropneumoniae.

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope ok the present invention in any way.

Deposits of strains Useful in Practicing the Invention

A deposit of biologically pure cultures of the following strains wasmade with the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. The accession number indicated was assigned aftersuccessful viability testing, and the requisite fees were paid. Accessto said cultures will be available during pendency of the patentapplication to one determined by the Commissioner to be entitled theretounder 37 CFR 1.14 and 35 USC §122. All restriction on availability ofsaid cultures to the public will be irrevocably removed upon thegranting of a patent based upon the application. Moreover, thedesignated deposits will be maintained for a period of thirty (30) yearsfrom the date of deposit, or for five (5) years after the last requestfor the deposit; or for the enforceable life of the U.S. patent,whichever is longer. Should a culture become nonviable or beinadvertently destroyed, or, in the case of plasmid-containing strains,lose its plasmid, it will be replaced with a viable culture(s) of thesame taxonomic description.

These deposits are provided merely as a convenience to those of skill inthe art, and are not an admission that a deposit is required under 35USC §S112. The nucleic acid sequences of these plasmids, as well as theamino sequences of the polypeptides encoded thereby, are incorporatedherein by reference and are controlling in the event of any conflictwith the description herein. A license may be required to make, use, orsell the deposited materials, and no such license is hereby granted.

    ______________________________________                                        Strain            Deposit Date ATCC No.                                       ______________________________________                                        HB101/pOM37/E1 (in E. coli)                                                                      4/7/92      68954                                          HB101/pSR213/E25 (in E. coli)                                                                   10/8/92      69083                                          ______________________________________                                    

C. Experimental

Materials and Methods

Enzymes were purchased from commercial sources, and used according tothe manufacturers' directions. Radionucleotides and nitrocellulosefilters were also purchased from commercial sources.

In the cloning of DNA fragments, except where noted, all DNAmanipulations were done according to standard procedures. See Sambrooket al., supra. Restriction enzymes, T₄ DNA ligase, E. coli, DNApolymerase I, Klenow fragment, and other biological reagents werepurchased from commercial suppliers and used according to themanufacturers' directions. Double stranded DNA fragments were separatedon agarose gels.

Bacterial Strains, Plasmids and Media

A. pleuropneumoniae serotype 1 strain AP37 and A. pleuropneumoniaeserotype 5 strain AP213 were isolated from the lungs of diseased pigsgiven to the Western College of Veterinary Medicine, University ofSaskatchewan, Saskatoon, Saskatchewan, Canada. A. pleuropneumoniaeserotype 7 strain AP205 was a Nebraska clinical isolate obtained from M.L. Chepok, Modern Veterinary Products, Omaha, Nebr. Other A.pleuropneumoniae strains were field isolates from herds in Saskatchewan.The E. coli strain HB101 (hsdM, hsdR, recA) was used in alltransformations using plasmid DNA. E. coli strains NM538 (supF, hsdR)and NM539 (supF, hsdR, P2cox) served as hosts for the bacteriophage λlibrary. The plasmids pGH432 and pGH433 are expression vectorscontaining a tac promoter, a translational start site with restrictionenzyme sites allowing ligation in all three reading frames followed bystop codons in all reading frames.

A. pleuropneumoniae strains were grown on PPLO medium (DifcoLaboratories, Detroit, Mich.) supplemented with 10 mg/ml β-nicotinamideadenine dinucleotide (Sigma Chemical Co., St. Louis, Mo.). Platecultures were incubated in a CO₂ -enriched (5%) atmosphere at 37° C.Liquid cultures were grown with continuous shaking at 37° C. without CO₂enrichment.

Iron restriction was obtained by adding 2,2'-dipyridyl to a finalconcentration of 100 μmol. E. coli transformants were grown in Luriamedium (Sambrook et al., supra) supplemented with ampicillin (100 mg/l).Transcription from the tac-promoter was induced by the addition ofisopropylthioglactopyranoside (IPTG) to a final concentration of 1 mmol.

Preparation and Analysis of Culture Supernatants, Outer Membranes andProtein Aggregates.

Culture supernatants, outer membranes, and aggregated protein wereprepared as previously described (Gerlach et al., Infect. Immun. (1992)60:892-898; Deneer, H. G., and Potter, A. A., Infect. Immun. (1989)57:798-804). Culture supernatants were mixed with two volumes ofabsolute ethanol and kept at -20° C. for 1 h. Precipitates wererecovered by centrifugation and resuspended in water. Outer membraneswere prepared by sarkosyl solubilization as previously described (Deneerand Potter, supra). For the preparation of protein aggregates, brothcultures (50 ml) in mid log phase (OD₆₆₀ of 0.6) were induced by theaddition of 1 mmol isopropylthiogalactoside (IPTG; final concentration).After 2 hours of vigorous shaking at 37° C., cells were harvested bycentrifugation, resuspended in 2 ml of 25% sucrose, 50 mmol Tris/HClbuffer pH 8, and frozen at -70° C. Lysis was achieved by the addition of5 μg of lysozyme in 250 mmol Tris/HCl buffer pH 8 (5 min on ice),addition of 10 ml detergent mix (5 parts 20 mmol Tris/HCl buffer pH 8 (5min on ice), addition of 10 ml detergent mix (5 parts 20 mmol Tris/HClbuffer pH 7.4, 300 mmol NaCl, 2% deoxycholic acid, 2% NP-40, and 4 partsof 100 mmol Tris/HCl buffer pH 8, 50 mmol ethylenediamine tetraaceticacid, 2% Triton X-100), and by sonication. Protein aggregates wereharvested by centrifugation for 30 min at 15,000 g. Aggregate proteinwas resuspended in H₂ O to a concentration of 5-10 mg/ml and solubilizedby the addition of an equal volume of 7 molar guanidine hydrochloride.The concentration of protein in the aggregate preparations wasdetermined by separating serial dilutions of the protein using SDS-PAGE.The intensity of the Coomassie blue stained bands was compared withthose of a bovine serum albumin standard (Pierce Chemical Co., Rockford,Ill.).

Western Blotting

Whole cell lysates of A. pleuropneumoniae grown in broth underiron-restricted conditions were separated by SDS-PAGE and electroblottedonto nitrocellulose membranes essentially as described by Towbin et al.(Towbin et al., Proc. Natl. Acad. Sci. U.S.A. (1979) 76:4350-4354).Nonspecific binding was blocked by incubation in 0.5% gelatine inwashing buffer (150 mmol saline, 30 mmol Tris-HCl, 0.05% Triton-X100).Antibody and alkaline phosphatase conjugate (Kirkegaard & PerryLaboratories, Inc., Gaithersburg, Md.) were added in washing buffer, andeach incubated for 1 h at room temperature. Blots were developed with asubstrate containing 5-bromo-4-chloro-3-indolyl phosphate (BCIP) andnitro blue tetrazolium (NBT) (ImmunoSelect, BRL, Gaithersburg, Md.) in100 mmol Tris/HCl buffer pH 9.5, 50 mmol NaCl, 5 mmol MgCl₂.

Preparation of Antisera

Serum against an A. pleuropneumoniae culture supernatant was obtained asfollows. A. pleuropneumoniae serotype 1 culture supernatant wasprecipitated with trichloroacetic (TCA; vol/vol), emulsified withincomplete Freund's adjuvant, and used to immunize rabbits twice atthree-week intervals. Porcine convalescent sera were obtained from pigsexperimentally infected intranasally by aerosol with A. pleuropneumoniaeserotype 1 strain AP37.

Preparation of DNA and Southern Blotting

Genomic DNA was prepared by SDS-facilitated freeze-thaw induced lysis asdescribed previously (Stauffer, G. V., et al., Gene, (1981) 4:63-72).Plasmid DNA was prepared from 100 μg/ml chloramphenicol-amplifiedcultures by alkaline lysis and cesium chloride-ethidium bromide gradientcentrifugation previously described (Sambrook et al., supra).

Restriction endonuclease digests were done in T4 DNA polymerase buffer(Sambrook et al., supra) supplemented with 1 mmol dithiothreitol and 3mmol spermidine. Digested DNA was separated on 0.7% agarose gels andtransferred onto nitro cellulose by capillary blotting. [³² P]-labelledprobes were prepared by random priming (Feinberg, A. P., and Vogelstein,B. (1983) Anal. Biochem. 132:6-13), and unincorporated nucleotides wereremoved by passage through a Sephadex G-50 column. Filters wereprehybridized in 5× Denhardt's solution-6× SSC (1× SSC is 0.15 mol NaCl,0.015 mol sodium citrate (pH 8))-0.5% SDS at 65° C. Filters werehybridized in the same solution at 55° C. and washed at 55° C. in 3×SSC-0.5% (low stringency), or at 65° C. in 0.1× SSC-0.5% SDS (highstringency).

Preparation and Screening of the A. pleuropneumoniae Serotype 1Expression Library

Genomic DNA from A. pleuropneumoniae AP37 was partially digested withthe restriction endonuclease Sau3AI. Fragments of 3000 Bp to 8000 Bpwere isolated by sucrose density gradient centrifugation (Sambrook etal., supra) and ligated into the BamHI and BglII sites of the expressionvectors pGH432 and pGH433, thus allowing for fusions in all threereading frames. E. coli HB101 was transformed and plated at a density ofapproximately 400 colonies per plate. Colonies were replica-plated ontonitrocellulose disks, induced for 2 h with 1 mmol IPTG, and lysed inchloroform vapor. Non-specific binding was blocked with 0.5% gelatin inthe washing buffer and, after removal of the cellular debris, themembranes were incubated with rabbit serum raised against the A.pleuropneumoniae AP37 culture supernatant and developed using goatanti-rabbit conjugate and substrate as described above.

Transposon Mutagenesis

The transposon TnphoA, carried by a lamba phage, as well as the alkalinephosphatase-negative E. coli strain CC118, were provided by J. Beckwith,Harvard Medical School, Boston, Mass. The mutagenesis was performed aspreviously described (Manoil, C., and Beckwith, J. (1985) Proc. Natl.Acad. Sci. U.S.A. 82:8129-8133) and the nucleotide sequence at theinsertion site was determined using an oligonucleotide primercomplementary to the first 20 bases of the phoA-gene in TnphoA (Chang etal. (1986) Gene 44:121-125; Manoil and Beckwith, supra).

Nucleotide Sequence Analysis

DNA sequencing was performed using M13 vectors and the dideoxy chaintermination method essentially as described (Sanger, F., et al. (1977)Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467). Nested deletions wereprepared by exonuclease III treatment (Henikoff, S. (1987) Methods inEnzymology 55:156-165). Specific primers were synthesized using thePharmacia Gene Assembler (Pharmacia Canada Ltd., Baie D'Urfe, Quebec,Canada). Both strands were sequenced in their entirety. The open readingframe (ORF) of the omlA gene was confirmed by TnphoA insertionmutagenesis as described above. The sequence was analyzed using theIBIPustell program and the GenBank database.

Primer Extension Mapping

A was prepared from A. pleuropneumoniae AP37 essentially as described byEmory and Belasco (Emory, S. A., and Belasco, J. G. (1990) J. Bacteriol.172:4472-4481). Briefly, 25 ml of bacterial culture (OD₆₆₀ =0.4) wascooled on crushed ice and centrifuged. The bacterial pellet wasresuspended in 250 μl of 10% sucrose, 10 mM sodium acetate (pH 4.5), andfrozen at -70° C. The pellet was thawed by mixing with an equal volumeof hot (70° C.) 2% SDS, 10 mM sodium acetate (pH 4.5). Then, 375 μl ofhot (70° C.) H₂ O-equilibrated phenol was added, the tubes werevortexed, frozen at -70° C., and spun for 10 min in an Eppendorfcentrifuge. The clear supernatant was removed, 2.5 volumes of ethanolwas added, and the RNA was stored at -70° C. until needed. The primerextension was done as described previously using a primer complementaryto a sequence within the ORF. 7-Deaza-dGTP and AMV-reverse transcriptasewere employed in order to prevent compressions.

Intrinsic Radiolabelling with [³ H]-Palmitic Acid, Immunoprecipitationand Globomycin Treatment

Labelling was done essentially as described previously (Ichihara, S. etal. (1981) J. Biol. Chem. 256:3125-3129). Briefly, [9,10-³ H] palmiticacid with a specific radioactivity of 55 Ci/mmol in toluene (AmershamCorp., Arlington Heights, Ill.) was lyophilized and dissolved inisopropanol to a concentration of 5 mCi/ml. A. pleuropneumoniae AP37 (inPPLO-broth) and E. coli transformants (in Luria broth containing 1 μmolIPTG were grown with methanol, and an immunoprecipitation analysis wasperformed essentially as previously described (Huang, et al. (1989) J.Bacteriol. 171:3767-3774). The OmlA-specific serum was obtained fromimmunized pigs, and protein G-Sepharose was used to recover theOmlA-porcine antibody complexes. The immunoprecipitated proteins wereresuspended in SDS-sample buffer, heated to 80° C. for 5 min andseparated by SDS-PAGE. The gels were fixed, treated with Amplify(Amersham Corp., Arlington Heights, Ill.), dried and exposed to X-rayfilm. Globomycin was dissolved in 50% dimethylsulfoxide at aconcentration of 10 mg/ml. This solution was added to an A.pleuropneumoniae AP37 culture grown to an OD₆₆₀ of 0.6 to a finalconcentration of 100 μg/ml. and growth was continued for 1 hour. Cellswere pelleted, resuspended in sample buffer and analyzed by SDS-PAGE andelectroblotting onto nitrocellulose, as described above, using theOmlA-specific serum.

EXAMPLES Example 1 Cloning and Expression of the A. pleuropneumoniaeSerotype 1 omlA Gene

An expression library of A. pleuropneumoniae strain AP37 serotype 1 inthe vector pGH432 lacI was screened with rabbit polyclonal antiserumgenerated against a concentrated culture supernatant of A.pleuropneumoniae by a colony immunoblot assay as described above.Colonies reacting with serum raised against the culture supernatant weresubcultured, induced with IPTG, and examined in a Western blot usingporcine convalescent serum. From among those clones which reacted in thecolony immunoblot assay, one clone which also reacted with convalescentserum was selected for further study. The E. coli transformant produceda protein which co-migrated with an immunoreactive protein from A.pleuropneumoniae AP37, and had an electrophoretic mobility of 50 k Da.Upon IPTG induction, this transformant produced the immunoreactiveprotein in aggregated form. The plasmid encoding this antigen wasdesignated as pOM37/E1 (ATCC Accession No. 68954), and the protein wasdesignated as OmlA.

Physical mapping showed that the plasmid contained a 5,000 Bp insert.Several deletion derivatives were constructed, and it was observed thattransformants containing the deletion derivative pOM37/E17 produced atruncated protein, thus indicating that the encoding gene overlaps theKpnI restriction enzyme site.

The nucleotide sequence of the gene encoding OmlA from pOM37/E1 is shownin FIG. 1 (SEQ ID NO: 1). The sequence was determined by dideoxysequencing of overlapping deletions generated by exonuclease IIIdigestion. The nucleotide sequence has one long open reading frame (ORF)starting at nucleotide position 158 and ending at position 1252. Theamino acid sequence of this open reading frame is also shown in FIG. 1(SEQ ID NO: 1). The predicted polypeptide has a molecular weight of39,780, with a consensus sequence for lipid modification at amino acidresidue 20. In order to confirm this, cells were labelled with [³H]-palmitate and immunoprecipitated with rabbit antisera generatedagainst the recombinant protein as described above. Followingpolyacrylamide gel electrophoresis and autoradiography, one band with anapparent molecular weight of 50,000 was observed, indicating that lipidmodification of the polypeptide had occurred. Further, when globomycinwas added, no [³ H]-palmitate-labelled material was visible on theautoradiogram. Globomycin is a specific inhibitor of signal peptidaseII. Thus, the omlA gene product is a lipoprotein. This may explain whyit migrates on polyacrylamide gels with an apparent molecular weight of50,000 when the predicted value is less than 40,000.

Immunoreactive product was expressed in transformants even in theabsence of IPTG induction. This suggests that a promoter recognizable byE. coli was located on the A. pleuropneumoniae-derived DNA upstream ofthe ORF. The simultaneous inducibility by IPTG, as well as the truncatedpolypeptide produced by E. coli pOM37/E17 transformants, indicated thelocation of the carboxy-terminal of the omlA gene as well as itsdirection of transcription.

Example 2 Analysis of Plasmid pOM37/E16

Colonies reacting with serum raised against the culture supernatant weresubcultured, induced with IPTG, and examined in a Western blot asdescribed in Example 1. The smallest plasmid expressing the full-lengthOmlA protein was designated pOM37/E16. Nucleotide sequence analysis ofpOM37/E16 revealed one ORF of 1083 Bp in length coding for a proteinwith a predicted molecular mass of 39,780 Da. It was preceded by aShine-Dalgarno consensus sequence AAGGAA 8 Bp upstream of the methioninecodon. The protein encoded by the nucleotide sequence of pOM37/E16 isidentical to that shown in FIG. 1 (SEQ ID NO: 1).

The first 19 amino acids of the polypeptide have the characteristics ofa lipoprotein signal peptide with a predicted cleavage site in front ofthe cysteine residue at position 20. The ORF was confirmed by twoindependent TnphoA-insertions 50 bp and 530 bp downstream from themethionine codon which, upon transformation of the phoA-negative E. colistrain CC118, gave rise to alkaline phosphatase-positive transformants.A GenBank data base homology search using the predicted amino acidsequence of OmlA did not reveal likely similarities (>35%) to known ORFsor polypeptides.

The primer extension located the beginning of the mRNA at a T-residue 76Bp upstream of the methionine start codon. The -10 and -30 regions areboth AT-rich, and the promoter-structure matches the E. coli consensuscharacteristics.

One of the TnphoA-insertions was found to be located within the signalpeptide. The expression of a functional PhoA protein in this fusion isprobably due to its location behind the hydrophobic core of the signalpeptide. The transcriptional start site as determined by primerextension analysis is preceded by a -10 and -30 region similar to thosecommon in E. coli promoters, Rosenberg, M., and Court, D., (1979) Annu.Rev. Genet. 13:319-353, and this finding is in accordance with theexpression found in non-induced E. coli transformants. Downstream of theORF, a palindromic sequence of 26 bp in length is present which mightact as a terminator sequence. Adhya, S., and Gottesman, M., (1978) Annu.Rev. Biochem. 47:967-996.

The predicted signal peptide cleavage site resulting in anamino-terminal cysteine residue of the mature protein was confirmed bylabelling of the E. coli transformants with [¹⁴ C]-palmitate andsubsequent immunoprecipitation using porcine anti-OmlA serum. Inaddition, it was shown that growth of A. pleuropneumoniae AP37 in thepresence of globomycin inhibited the palmitate-labelling of OmlA as wellas the processing of the OmlA precursor protein.

The expression of the OmlA protein was independent from the level ofiron in the growth medium. The protein was present in whole membranes,outer membranes as prepared by sucrose gradient centrifugation, andmembrane blebs; it was absent in sarcosyl-treated outer membranes and inhigh-speed supernatants.

Example 3 Cloning, Expression and Sequencing of the A. pleuropneumoniaeSerotype 5 omlA Gene

Genomic DNA from A. pleuropneumoniae serotype 5 strain AP213 wasdigested to completion with StyI and ligated into the NcoI site of thepGH432 lacI-derivative, pAA505. HB101 recombinants were screened withconvalescent serum obtained from a pig which had been infected with A.pleuropneumoniae serotype 5. One positive clone, HB101/pSR213/E1, wasselected for further analysis. HB101/pSR213/E1 was shown to containthree StyI fragments. In order to isolate the DNA coding for theimmunoreactive protein, StyI fragments from this plasmid were treatedwith DNA polymerase I Klenow fragment to fill in the 5' extensions.These fragments were ligated into the SmaI site of the vector,pGH432/lacI. A seroreactive clone, designated HB101/pSR213/E4, wasisolated and shown to produce a seroreactive protein with an apparentmolecular weight of 50 kDa. However, the protein was not expressed athigh levels. To increase the level of expression, plasmid pSR213/Er wasdigested with BglII (which cuts the vector sequence upstream of thegene) and then partially digested with AseI (which cuts at the beginningof the coding region of the gene). The 5' extensions were filled in withDNA polymerase I Klenow fragment, and the plasmid recircularized byligation. The resulting clone, HB101/pSR213/E25 (ATCC Accession No.69083), overexpressed the seroreactive protein.

Both strands of the A. pleuropneumoniae serotype 5 omlA gene weresequenced using M13 vectors as described above. The nucleotide sequenceand predicted amino acid sequence are shown in FIG. 2 (SEQ ID NO: 2).The open reading frame shown in the figure codes for a protein similarto the omlA product of A. pleuropneumoniae serotype 1, showingapproximately 65% identity at the amino acid level. Thus, the openreading frame present in pSR213/E25 codes for the serotype 5 equivalentof omlA.

Example 4 Distribution of the QmlA gene in the A. pleuropneumoniae typestrains.

Genomic DNA from all 12 A. pleuropneumoniae type strains was analyzed ina Southern blot using the A. pleuropneumoniae AP37-derived omlA-gene asprobe. The StyI-restricted DNA from all A. pleuropneumoniae type strainsreacted with the probe under low stringency conditions, and the DNA fromserotypes 1, 2, 8, 9, 11, and 12 remained hybridized to the probe underhigh stringency washing conditions.

Whole cell lysates from all A. pleuropneumoniae type strains, grownunder iron-restricted conditions, were analyzed in a Western blot usingthe serum from pigs immunized with the recombinant OmlA protein. Thesame strains that hybridized to the DNA probe under high stringencywashing conditions bound the anti-OmlA sera, and the whole cell lysatesfrom the A. pleuropneumoniae type strains for serotypes 1, 9, and 11reacted more strongly than those of serotypes 2, 8, and 12.

Example 5 The Protective Capacity of Serotype 1 OmlA Recombinant Protein

The OmlA protein was prepared from E. coli HB101/pOM37/E1 byIPTG-induction of a log phase culture followed by cell harvest anddisruption, and separation of the inclusion bodies by centrifugation.The inclusion bodies were solubilized with guanidine hydrochloride andmixed with Emulsigen Plus (MVP Laboratories, Ralston, Neb.) and salineso that the final protein concentration was 0.5 μg/ml, 2.5 μg/ml or 12.5μg/ml. Groups of 7 pigs were vaccinated with 2 ml of the vaccines or aplacebo containing Emulsigen Plus but no protein. Each group wasrevaccinated 21 days later and finally challenged 7 days after the boostwith an aerosol of A. pleuropneumoniae (serotype 1). Clinical signs ofdisease were followed for 3 days, and 7 days after challenge allsurvivors were euthanized. The significance of the difference inmortality rates among the different groups was determined using a G²likelihood ratio test (Dixon, W. J., et al., BMDP Statistical SoftwareManual, University of California Press, 1988, pp. 229-273.) The resultsare summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Protective Capacity of OmlA Against Challenge                                 with Actinobacillus pleuropneumoniae serotype 1.                                     MORTALITY     CLINICAL SCORE                                           GROUP    Day 1   Day 2   Day 3 Day 1 Day 2 Day 3                              ______________________________________                                        Placebo  0/7     7/7     7/7   2.86  3.00  --                                 OmlA-1 μg                                                                           0/7     0/7     0/7   1.21  1.00  0.93                               OmlA-25 μg                                                                          0/7     1/7     1/7   1.14  0.86  0.58                               ______________________________________                                    

Within 2 days of challenge, all of the pigs which received the placebowere dead while only 1 of the OmlA-vaccinates had died. Clinical signsof disease were significantly lower in the vaccinates on day 1post-challenge, the only day on which a comparison could be made due tohigh mortality in the placebo group. Thus, the omlA gene product of A.pleuropneumoniae (serotype 1) is an effective immunogen for theprevention of porcine pleuropneumonia caused by A. pleuropneumoniae .Immunization of pigs with the recombinant OmlA protein induced a strongimmune response and significantly lowered mortality. These resultsdemonstrate that protection against A. pleuropneumoniae serotype 1 canbe achieved by immunization with a single protein antigen. Since therecombinant protein used for the vaccination trial was produced as anaggregate in E. coli, the lipid modification does not appear to benecessary for the induction of a protective immune response.

Example 6 The Protective Capacity of Sterotype 5 OmlA RecombinantProtein

OmlA protein was prepared from HB101/pSR213/E25 and formulated withEmulsigen Plus as described in Example 5 so that each 2 ml dosecontained 25 μg of protein. Pigs were vaccinated, boosted and challengedwith A. pleuropneumoniae serotype 5 strain AP213 as described in Example5. The results shown in Table 2 indicate that vaccination with OmlA fromserotype 5 reduced morbidity, mortality and lung damage associated withActinobacillus pleuropneumoniae infection. It is predicted thatvaccination with both serotype I and serotype 5 OmlA proteins wouldprotect pigs against infection with all A. pleuropneumoniae serotypes,with the possible exception of serotype 11.

                                      TABLE 2                                     __________________________________________________________________________    Protective Capacity of OmlA Against Challenge with                            Actinobacillus pleuropneumoniae serotype 5.                                                MEAN BODY TEMP.                                                                           MEAN                                                              (°C.)                                                                              CLINICAL SCORE                                       GROUP                                                                              MORTALITY                                                                             Day 1                                                                             Day 2                                                                             Day 3                                                                             Day 1                                                                             Day 2                                                                             Day 3                                                                             LUNG SCORE                               __________________________________________________________________________    Placebo                                                                            3/3     40.87                                                                             40.40                                                                             41.00                                                                             1.33                                                                              1.58                                                                              2.13                                                                              O                                        OmlA 0/4     39.67                                                                             39.65                                                                             39.73                                                                             0.25                                                                              0.44                                                                              0.31                                                                              ND                                       __________________________________________________________________________

Thus, subunit vaccines for use against A. pleuropneumoniae aredisclosed, as are methods of making and using the same. Althoughpreferred embodiments of the subject invention have been described insome detail, it is understood that obvious variations can be madewithout departing from the spirit and the scope of the invention asdefined by the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1340 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A ) NAME/KEY: CDS                                                            (B) LOCATION: 158..1252                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GATCGGCTTTTACAGCGATTGCAGAATGATTGAATTGTAAACTTTAGAGCTTTATATTTT60                GTTTAATGGTATTATATTTACTTATATTTATGATTCTTAGTTTTTATTGTAAATTAAAGT120               GTTTATTTAT TGTATTTTAAGTATAAGGAATTTTTTAATGAATATTGCAACAAAATTAAT180              GGCTAGCTTAGTCGCTAGTGTAGTGCTTACCGCATGTAGTGGCGGCGGCTCATCGGGTTC240               ATCGTCTAAACCAAATTCGGAACTTACACCTAAGGTTGATATGTCCGCACCAAAAG CGGA300              GCAGCCAAAAAAAGAGGAAGTTCCACAAGCGGATAATTCGAAAGCGGAAGAACCAAAAGA360               GATGGCTCCGCAAGTAGATAGCCCGAAAGCGGAAGAACCAAAAAATATGGCTCCACAAAT420               GGGTAATCCAAAACTAAATGACCCACAAGTAAT GGCTCCGAAAATGGATAATCCGCAAAA480              AGATGCCCCAAAAGGAGAAGAACTAAGTAAGGATAAAAGTAATGCGGAAATTCTTAAGGA540               ATTAGGGGTTAAGGATATTAATTCAGGTATCATTAATAATGCTGATGTAGTTCTGAATTT600               AAAAATAGAT GAAAAAGATCACATTACAGTCGTATTAGATAAGGGTAAGATTAATCGTAA660              TCATCTAAAAGTAACTAATACAATTTCTGCTCAAGACATTAAAACCTTAAAAGATTCTTC720               AGGCAAATTGTTGGGTTACTATGGATATATGCAGTTAAATCAAGTTCGACAAGATG AAAA780              TTATAGCGATGAAAAAGTTAGTTTGAATGAATATTATTTATTATCAATGAACGATGCCGA840               TAAAATACGTCCGACTAAATCTATATCATATAAGGGAGACATGTTTTATAGTTACAAAGA900               TGTAGGAAATCAGAAATTAAAGGCTTCTGTAGA AGCTTCTTATGATGATGTAACAAAAAA960              AGTATCAATGAAAGTATTTGGTGAGAATAATGATTACTGGAAATTAGGTGAGTTTGGTAG1020              AACTAATTTATTAGAAAATCAAGTGACTGGAGCAAAAGTTGGCGAAGATGGTACCATTAT1080              AAATGGAACT TTATATTCTAAAATAGATAATTTTCCTTTAAAACTAACTCCTGACGCAAA1140             CTTCTCTGGGGGTATTTTCGGTAAAAATGGCGAAGTATTAGCCGGAAGTGCTATTAGTGA1200              AAAATGGCAAGGCGTAATCGGTGCTACGGCAACCACAAAAGAAGATAAAAAATAAA CGCT1260             TTGCTAACTAAACCAAAAGTTATCCTTCGGGATAGCTTTTTTACTTTTTAATCAGACCTA1320              ATAGTGCATCGGTAAAAGAT1340                                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A ) LENGTH: 2398 base pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 293..1393                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TCTAGAAAATGCACTGACAAAATTAGGACTTTCAGTGCGTGCCTATCATAGGATTTTA AA60               AGTTTCCCGCACAATTGCCGATTTGGCGAACGAACCGAATATTCAACAAATCCATCTTGC120               CGAAGCGTTAGGTTATCGAGCGATGGATCGGCTTTTGCAGAGGTTGCAGAACGATTAAAT180               TGTAAACTTTAGAGCTTTATATTTTGTTTGATGGT ATTATATTTATGATTTTTAGTTTTT240              ATTGTAAATTAAAGTGTTTATTTATTGTATTTTAAGTATAAGGAATTTTTTAATGAATAT300               TGCAACAAAATTAATAGCCGGTTTAGTCGCAGGTTTAGTGCTTACCGCATGTAGTGGCGG360               CGGCTCATCGGG TTCATCGCCTAAACCAAATTCGGAATCTACGCCTAAGGTTGATATGTC420              CGCACCAAAAGCGGAGCAGCCAAAAAAAGAGGAAGCTCCGCAAGCGGATAGCCCGAAAGC480               AGAAAAACCAAAAAGTATTGCTCCACTGATGATGGAAAACCCAAAAGTAGAGAAACAG AA540              AGAAAATAACCTACAAGAGAAAAGTCCAAAGGCAGACGAACCGCAAGTAATGGATCCAAA600               ATTAGGTGCTCCACAAAAAGATGATCAGAAGTTAGAAGAACCTAAGAATAAAAGTAATGC660               GGAAATTCTTAAGGAATTAGGGATTAAGGATATTA CTTCAGGGACAATTAGTATTTCCGA720              TATTGAATTGAATCTACAATTAGATAGCAATGATAATGTGAAAATATCTTTGTTAAATGA780               GAATTTAATGCGTGATAATTTAACGATTAATAATAAGATTGCAGGTTCGGATATTAGAAC840               GTTAAAAGATTC TTCAGGTAGATTGTTAGGTTATTATGGTTATGTGCAATTGAATCAAGT900              TACACAAGACTCTCGTGACCCAGATAATTATAAGCATCAGTTTGAAAATCATTATTTACT960               GTCTATGAATGATGCTGAGAAAATATTACCAGAAAAGTCGTTAGAATATAAAGGTAGT AT1020             GATTTACGGATATAATACTTCTGGAAATGAAAAGCTTACTGCAGAAGTGAATGCTAAATA1080              TGATAGTTCAACTAAAAAATTATCAATGAAAGTATATGATAATGATCGTTATTGGAAATT1140              AGGCGAAGTAATGAGTAACAATGTTAGATTACCAG AAGAAAAAGTTGATGGTGTGAAAGT1200             TGATTCTGACGGAACAATTAATGCTCGTTTATATTTAAGCACTGAAGAACCATTAAAATT1260              AACCCCTGACGCCAATTTCTCCGGTGGTATTTTTGGGAAAAACGGTGAAGTACTGGCAGG1320              AAAAGCGGAAAG CATTAAGGGAGAATGGCAAGGCGTAATCGGTGCTACGGCAACAACAAA1380             AGAAGATAAAAAATAACGCTTTGCTTACCAAACTAAAAGCTATCCTTCGGGATAGCTTTT1440              TTACTTTTTAATCAGTGCCAATAGTGCATCGGTAAAAGATTCCGGGTTTTCATAATGT GC1500             GTTATGTCCGGCATTAGGAATAAGCTGATGATGAAGTTTATTATCGGAGACGATTTTTCT1560              AAATTTCCGATCATATTCGCCGATCAAAAAAGTGATAGTCTGCCGAGCTTCGGAGAGCTG1620              CGGTAAAAAATAAGGTTGCTTTGCAAGACTAGTCG CTTCAAGCATAGCCGCAACAACTGA1680             TCCGTTATTGTTTTGCGCCGAAAAACGATTAAATTTGGACCGCTTGTGTTGGTCTAAATT1740              GGCAAAAACGGCTTGTTGATACCAATCATTTAATACTTTCACTATCGGTTCGTTACGGAA1800              ACGTTTCGCCCA TTGATGGTCGTTTTGCCAACGAGCTTGGCGTTCCTCATCTGTTGCTAA1860             GCCGATGTTCGCTCCTTCAAGAATCGTATGTTTTAGCTGAGGATTATTGGCATTGAGCGC1920              ATAGTCAACGCTAAACGCCCGCCTAACGAATAGCCGACCAAATAAAAAGGCTGATTGC CG1980             ATATAATGCAGAACGGTTTGATGAATCAATTCTCTCGTGTGGGAAAAGCCGTAGCAGGGG2040              ATATGTTCGCTTGCCGCCATGCAGAGGAAGGTCAATGGTAAGCGGTCGAATTTGCGGAAA2100              ANNNNNCTAGCACCGCTTGCCAAATCTTGTTGCGA ACCGAGTAAACCGTGCAGGAAAAAA2160             CCACCGGCATACCCGTTTCACGATGCCATGTTGCGTGGAGCATTAGGCAATTTCCGCTTG2220              TGAGATTTGTTTAACTAAGGATTTGTAAAGATTGCTACCGTCTTGATCGTTCACTTTAAT2280              TTCAACGCATAG TCACGCCTTTACGTCCGTAAGCGAGTTTCAGTTTCGCTTTCAGATCGG2340             CCCAAGTAAACGGACGGATATATTCAATGCCGAATATGGTCGCAATCGGTGCGAATTC2398            

We claim:
 1. A purified, Actinobacillus pleuropneumoniae outer membrane protein, wherein the protein is an immunogenic serotype 1 Actinobacillus pleuropneumoniae outer membrane lipoprotein A comprising an amino acid sequence as depicted in FIG. 1 (SEQ ID NO: 1).
 2. A purified, Actinobacillus pleuropneumoniae outer membrane protein, wherein the protein is an immunogenic serotype 5 Actinobacillus pleuropneumoniae outer membrane lipoprotein A comprising an amino acid sequence as depicted in FIG. 2 (SEQ ID NO: 2).
 3. A vaccine composition comprising a pharmaceutically acceptable vehicle and an immunogenic serotype 1 Actinobacillus pleuropneumoniae outer membrane lipoprotein A comprising an amino acid sequence as depicted in FIG. 1 (SEQ ID NO: 1).
 4. The vaccine composition of claim 3 further comprising an adjuvant.
 5. A method of treating or preventing an Actinobacillus pleuropneumoniae infection in a vertebrate subject comprising administering to said subject a therapeutically effective amount of a vaccine composition according to claim
 3. 6. A method of treating or preventing an Actinobacillus pleuropneumoniae infection in a vertebrate subject comprising administering to said subject a therapeutically effective amount of a vaccine composition according to claim
 4. 7. A vaccine composition comprising a pharmaceutically acceptable vehicle and an immunogenic serotype 5 Actinobacillus pleuropneumoniae outer membrane lipoprotein A comprising an amino acid sequence as depicted in FIG. 2 (SEQ ID NO: 2).
 8. The vaccine composition of claim 7 further comprising an adjuvant.
 9. A method of treating or preventing an Actinobacillus pleuropneumoniae infection in a vertebrate subject comprising administering to said subject a therapeutically effective amount of a vaccine composition according to claim
 8. 10. A method of treating or preventing an Actinobacillus pleuropneumoniae infection in a vertebrate subject comprising administering to said subject a therapeutically effective amount of a vaccine composition according to claim
 7. 11. A vaccine composition comprising:(a) a pharmaceutically acceptable vehicle; (b) an immunogenic serotype 1 Actinobacillus pleuropneumoniae outer membrane lipoprotein A comprising an amino acid sequence as depicted in FIG. 1 (SEQ ID NO: 1); and (c) an immunogenic serotype 5 Actinobacillus pleuropneumoniae outer membrane lipoprotein A comprising an amino acid sequence as depicted in FIG. 2 (SEQ ID NO: 2).
 12. The vaccine composition of claim 11 further comprising an adjuvant.
 13. A method of treating or preventing an Actinobacillus pleuropneumoniae infection in a vertebrate subject comprising administering to said subject a therapeutically effective amount of a vaccine composition according to claim
 12. 14. A method of treating or preventing an Actinobacillus pleuropneumoniae infection in a vertebrate subject comprising administering to said subject a therapeutically effective amount of a vaccine composition according to claim
 11. 